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Excretion of steroids by the newborn infant
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71,
91-99
(1957)
Excretion of Steroids by the Newborn Infant’ Helen Strauss Hirs~h,~ David L. Berliner and Leo T. Samuels From the Departments of Biological Chemistry and Anatomy, University Utah College of Medicine, Salt Lake City, Utah Received
December
of
11, 1956
INTRODUCTION
Both chemical and histologic evidence indicates that there is a marked change in the steroid metabolism of the human being during the adjustment from intrauterine to independent existence. The involution of the fetal adrenal after birth is a well-documented phenomenon (1, 2) ; and Migeon et al. (3) have shown that the level of dehydroepiandrosterone in cord blood is higher than in the blood of the mother, but that it falls below measurable levels within a few days after birth. He concludes that the high levels of the steroid in the cord blood probably originate from the fetal rather than the maternal adrenal, and that the decrease is associated with the adrenal involution. Gardner (4, 5) has reported high levels of 17-ketosteroids in the plasma of newborn infants; these fell below measurable levels in a few days. Eagle (6) has described the presence of dehydroepiandrosterone and similar substances in the urine of premature babies, and Ulstrom and Doeden (7) have found a series of peaks when the Zimmermann reaction was applied to fractions from a column chromatogram prepared by a modification of the method of Dingemanse et al. (8). No consideration, however, has been given to the nonketonic steroids. In the present study the daily excretion of steroids during the first 10 days of life was investigated. The antimony trichloride reaction of Pincus (9) decreased progressively over the first 10 days. From the pooled nonketonic fractions androstane-3cr, 17/?-diol was crystallized and identified. 1 This work was supported in part by research grants from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service and from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 2 Present address : Bethesda, Maryland. 91
92
HIRSCH,
BERLINER
AND
SAMUELS
METHODS
A. Collection of Specimens Because of the difficulties in obtaining quantitative specimens of urine from newborn infants, it was decided to use the entire excreta as collected in the diapers. This would also allow the collection of steroids excreted via the intestinal tract and, since the gut of the newborn infant contains relatively few bacteria, errors due to the action of organisms would not be great. Cotton diapers which had been laundered in the hospital laundry were extracted with chloroform to eliminate the extraneous lipide material and then dried and sterilized. During each 24-hr. period each diaper, as soon as it was removed, was placed in a covered glass jar containing ethanol. The diapers were then allowed to stand in the ethanol for another 24 hr. with occasional agitation. At the end of this period the ethanol extract was drained as completely as possible from the cloths, and was replaced with butanol. This solvent was again left in contact with the diapers for another 24 hr. with occasional stirring and removed as before. A third extraction under the same conditions was then carried out with chloroform. The three extracts from each sample were combined and taken to dryness by vacuum distillation. The residue was dissolved in a small amount of chloroform and alcohol, 125 ml. of 4% sulfuric acid was added, and the mixture was hydrolyzed under reflux for 30 min. After cooling, the hydrolyzate was extracted four times with 50 ml. chloroform, the extracts were combined, and the solvent was removed by evaporation under vacuum. The residue was dissolved in ether and extracted with three portions of 2.5 N NaOH which was discarded. The ether layer was then washed once with an equal volume of 10% sulfuric acid and three times with equal volumes of distilled water. The extracts at the end of this procedure contained large amounts of lipide material and pigments, and removal of most of these impurities was essential for the subsequent analysis. The residue from the ether extract containing the neutral steroid fraction, therefore, was completely dissolved in a mixture of 2 parts of ether to 7 parts of acetone, and a small amount of saturated magnesium chloride solution in alcohol (approximately 30 drops/l0 ml. of ether-acetone mixture) was added. The sample was placed in the refrigerator for at least 8 hr. The solution was then filtered and the precipitate washed thoroughly with cold acetone. The ether-acetone solution was taken to dryness and the residue was distributed between 70% ethanol and pentane. The alcohol portion was extracted three times with equal volumes of pentane, and the combined pentane extract was then backwashed with one volume of 70% ethanol. The ethanol fractions were combined and the solvent was removed by evaporation; the pentane extract was discarded. The ethanol-soluble residue was then divided into ketonic and nonketonic fractions according to the procedure of Pincus and Pearlman (10) using Girard reagent T. Besides accomplishing the separation of ketonic and nonketonic steroids this procedure removed most of the remaining pigment from the samples. After evaporation of the ether, the ketonic and nonketonic residues usually consisted of small droplets of lipide material containing slight amounts of yellow pigment. Both the ketonic and nonketonic extracts were subjected to two color reactions: the Callow modification of the Zimmermann reaction (11,12) and the anti-
93
EXCRETION OF STEROIDS mony trichloride reaction of Pincus (9). Aliquots equal to one-fifth excretion were used for the color reactions unless otherwise specified. ing aliquots of each type of extract were then combined, and isolation steroids was attempted.
of the 24-hr. The remainof individual
RESULTS
After reactions with the Zimmermann reagent most ketonic extracts gave absorption curves which had maxima around 500 rnp while the nonketonic fractions showed only general decreasing absorption over the 400-700~pm range. As can be seen in Fig. 1, where the spectra for both ketonic and nonketonic fractions of three subjects are given, there was considerable background absorption, however, in all extracts. Typical results with the antimony trichloride reaction are shown in Fig. 2. All nonketonic fractions gave a green color with a maximum absorption around 600 rnp; this was absent from all ketonic samples. Of the various steroids tested by Pincus, the only nonketonic compound giving an absorption in this region was androstanediol; pregnanediol and cholesterol showed weak maxima at 680-690 rnp (9). Since reactions were obtained with both the ketonic and nonketonic Non-Ketonic Ketonic
400 FIG. 1. Absorption curves tracts from diaper collections Reaction.
500 WAVELENGTH
---
600
700
rnp
for ketonic and nonketonic fractions during the first 24 hr. after birth.
of three exZimmermann
94
HIRSCH,
BERLINER
AND
SAMUELS
Non-Ketonic Ketonic - - - - -
I
I
I
I
I
I
I
I
I
I
I
I
500
600 700 WAVELENGTH rnfi FIG. 2. Absorption curves for ketonic and nonketonic fractions of three extracts from diaper collections during the first 24 hr. after birth. SbCZ3Reaction.
extracts from excreta during the first 24 hr. of life, collections were continued over a IO-day period on two female infants. The results of the analyses, using the Allen correction for background absorption (13), are shown in Table I. Unfortunately, in the case of S.L. a number of samples were lost due to various factors, but the two series show the same general pattern. There is a tendency for the excretion of both types of compound to decrease. This is particularly striking in the case of the nonketonic substance which has practically disappeared by the tenth day. Because of the relatively strong reaction with antimony trichloride given by the early nonketonic fractions, the remaining portions of these fractions were pooled and an attempt was made to isolate and identify compounds therein. The pooled extract was separated into alcoholic and nonalcoholic fractions with succinic anhydride (10). Both the alcoholic and the nonalcoholic fractions were then chromatographed on aluminum oxide (Merck, according to Brockman). The chromatograms were developed with increasing proportions of benzene to petroleum ether, benzene, increasing concentrations of ethanol in benzene, and finally absolute ethanol. Most of the nonketonic, nonalcoholic fraction was eluted in the benzene-petroleum ether mixtures. No crystalline material was obtained
EXCRETION
OF
TABLE
of Androstane-$a,l7p-diol
Excretion
after 1
2 3 4 5 6 7 8 9 10
17-Kb
0.92 0.72 0.72 0.46” 1.19c
0.24 0.17 0.17 0.20” 0.40”
0.35 0.29 0.46 0.08
0.13 0.06 0.06
TABLE
of Nonketonic
Weight Fr%:?
of material
the First Baby
mg.
a Androstane-3cr, 17@-diol used as standard. b Dehydroepiandrosterone used as standard. c Probably portion of excretion from 4th day included
Chromatography
during
R.I.
mg. dial”
birth
I
and l’l-Ketosteroids Postnatal Days Baby
Days
95
STEROIDS
Alcoholic
Ten
S.L. mg.
mg. dioP
17-Kb
0.85 0.67
0.13 0.30
0.37 0.47
0.07 0.12
0.10
0.07
0.02
0.08
in collection
for 5th day.
II Fraction
put on the column:
Eluant
on Aluminum
Oxide
34.5 mg. Approx.
wt.
of fraction
m.
5 6 7 8 9 10 11 12
Benzene-petroleum ether 1: 1 Benzene Benzene 0.2 and 0.3’% alcohol in benzene 0.5, 0.7, 1 .O, and 2.0% alcohol in benzene 0.3% alcohol in benzene 4.0, 5.0, 7.0, and 10.0% alcohol in benzene 50.0y0 alcohol in benzene and absolute alcohol
2.6 1.2 3 .O (crystalline) 2.0 2.0 1.5 (crystalline) 2.0
in any fraction, and none of the fractions gave anything but background color, either with the Zimmermann reagent or antimony trichloride. As shown in Table II, two crystalline fractions were obtained from the nonketonic alcoholic material, one eluted with benzene (No. 7), and the other with 3% ethanol in benzene (No. 10). Fractions 7 and 10 were recrystallized from 70 % methanol. The supernatant solutions were drawn off and used for the antimony trichloride reaction, as well as the entire amounts of the other fractions. Fractions 5, 6, 12 and the supernatant from 7 gave only background color but fractions 8, 9, 11, and the super-
96
HIRSCH,
BERLINER
AND
SAMUELS
natant from 10 gave the typical green color observed in the diaper extracts; the supernatant from fraction 10 was particularly strong. The crystals of fraction 10 melted at 228°C. after recrystallization from dilute methanol. The acetate was prepared and melted at 160-162°C. These melting points correspond to that of androstane3cY) 17/?-diol, m.p. 223°C and its acetate, 162-163°C. (14). The free compound was then chromatographed on paper in the benzene-formamide system of Zaffaroni (15) where it had an R, of 0.21. This compound was eluted in methanol and divided into three aliquots for the following procedures: (a) chromogen determination with sulfuric acid (16)) (b) oxidation with chromic trioxide (17), and (c) acetylation with acetic anhydride-l-Cl4 (18). (a) The sulfuric acid chromogen showed maxima at 290, 340, and 410 rnp, identical with known androstane-&, 17/?-diol. (b) After oxidation the compound was chromatographed in the hexane-formamide system of Zaffaroni, where it showed an R, of 0.31, gave a positive Zimmermann reaction, and had a maximum of 290 mp in sulfuric acid, identical with known androstane-3,17-dione. (c) This aliquot was acetylated with acetic anhydride-l-C4 (specific activity, 2 mc./pmole) and pyridine overnight at room temperature. Acetylation was stopped by the addition of water, and the mixture was extracted with ethyl acetate. The ethyl acetate was evaporated, and at this time crystalline acetate originally prepared from the material isolated on the alumina column was added. The mixture was chromatographed on a paper immersed in a solution of methanol and formamide (2:s) in the heptane-formamide system at a room temperature of 4°C. The paper was dried and the compound identified by the microchemical isotopic technique described elsewhere (19). The compound had an R, of 0.77, identical with known androstan-3cy, 17p-diol-3,17-diacetate. A small amount of another radioactive contaminant with low polarity was found, which has not yet been identified. The radioactive androstan-3a, 17/3-dial-diacetate was hydrolyzed with 10 mg. of acylase using a 0.2 M phosphate buffer at pH 7.0 for 3 hr. at 37°C. The incubation was stopped by adding chloroform, and extracted. The chloroform extract was chromatographed in the benzene system, where the compound had an Rf of 0.23. This substance was further oxidized with chromic trioxide and run in the hexane-formamide system, where the compound had an Rf of 0.31, a positive Zimmermann reaction, and a maximum of 290 rnp in concentrated sulfuric acid, identical with
EXCRETION OF STEROIDS
IDENTIFICATION
97
OF ANDROSTAN-3~,17&diol.
FIG. 3. Method of identification of androstan-3a, 17&diol in chromatographic studies. (*) indicates position of CY4.
known androstan-3,17-dione. The evidence seems conclusive, therefore, that the crystalline nonketonic compound was androstane-3a, 17p-diol. The outline of the reactions used for identification is shown in Fig. 3. DISCUSSION
The evidence here presented indicates that one of the major (219 steroids excreted by the newborn infant is androstane-3cw,17@-diol. It appears to account for the reaction with antimony trichloride characteristic of the extracts during the first few days. Salhanick et al. found that radioactivity from testosterone-4-Cl4 did not cross the placental barrier, and that 4-androstene-3,17-dione was higher in cord blood than in the maternal circulation.3 Migeon et al. (3) also found that dehydroepiandrosterone conjugates were higher in the cord circulation. Apparently Cl9 compounds do not readily cross the placental barrier in either direction. Since the compound giving this reaction continues to be excreted during the first 10 days of life, it seems unlikely that it, or its precursor, entered the infant’s blood stream from the mother’s circulation. The more proba3 Salhanick, H. A., Jones, J. E., and Berliner, D. L., in preparation.
98
HIRSCH, BERLINER AND SAMUELS
ble hypothesis seems to be that the disappearance of the compound is associated with the involution of the fetal zone of the adrenal cortex. What the precursor of androstanediol would be is uncertain. It may arise from complete reduction of the 4-androstene-3,17-dione found in cord blood (20). While androstanediol is perhaps a minor product of testosterone metabolism (21), it does not appear to be a major steroid in the urine of adult human beings. Its presence in infancy may represent a difference in metabolism. In the adult liver the conjugation process is so rapid that the major portion of the steroids are combined with the acids as soon as the ketone group on C-3 is reduced to an alcohol, and relatively little undergoes the further reduction at C-17. If conjugation mechanisms are not fully developed in the liver of the newborn, a greater portion of Cl9 steroids may be reduced to the diols before combination with glucuronic or other acids. It is also possible that the diol may have been present in the intestinal contents, a source which has not been well examined in the adult. Further work will be needed to determine its origin. ACKNOWLEDGMENT The advice and assistance of Dr. Harold L. Mason, Mayo Clinic, Rochester, Minnesota, during the isolation and recrystallization procedures is gratefully acknowledged. SUMMARY
The entire excreta from babies during the first few days after birth were extracted and analyzed for neutral steroids related to hormonal function. Material giving the Zimmermann reaction was found in the ketonic fraction, but no reaction with the antimony trichloride reaction of Pincus was obtained. The nonketonic fraction gave the antimony trichloride reaction. Both the ketonic Zimmermann-positive and the nonketonic antimony trichloride-positive substances decreased to very low levels during the first 10 days after birth. The material responsible for the color reaction in the nonketonic fraction was identified as androstane-3a!, 17fi-diol, probably a metabolic product of compounds formed in the fetal adrenal cortex. REFERENCES 1. ELLIOTT, T. R., AND ARMOUR, R. G., J. Pathol. Bacterial. 16, 481 (1911). 2. KERN, H., Deut. med. Wochschr. 37, 971 (1911). 3. MIGEON,~. J., KELLER, A. R., AND HOLMSTROM,E. G., Bull. Johns Hopkins Hosp. 97, 415 (1955).
EXCRETION
4. GARDNER, L. 5. GARDNER, L. 6. EAGLE, J. F., 7. ULSTROM, R. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
OF STEROIDS
99
I., J. Clin. Endocrinol. and Metabolism 13, 941 (1953). I., AND WALTON, R. L., Helv. Paediat. Acta 4, 311 (1954). Proc. Sot. Exptl. Biol. Med. 81, 571 (1952). A., AND DOEDEN, D., “Adrenal Function in Infants and Children,” Chap. 3, p. 31. Grune and Stratton, New York, 1955. DINGEMANSE, E., HUIS IN'T VELD, L., AND HARTOGH-KATZ, S., J. Clin. Endocrinol. and Metabolism 12, 66 (1952). PINCUS, G., Endocrinology 32, 176 (1943). PINCUS, G., AND PEARLMAN, W. H., Endocrinology 29, 415 (1941). CALLOW, ?J. H., CALLOW, R. K., AND EMI\IENS, C. W., Biochem. J. 32, 1312 (1938). ZIMMERMANN, W., Z. physiol. Chem. 233, 257 (1935). ALLEN, W. M., J. Clin. Endocrinol. 10, 71 (1950). RUZICKA, L., GOLDBERG, M. W., AND MEYER, J., Helv. Chim. Acta 18, 994 (1935). ZAFFARONI, A., Recent Progr. Hormone Research 8, 51 (1953). ZAFFARONI, A., J. Am. Chem. Sot. 72, 3828 (1950). ZAFFARONI, A., AND BURTON, R. B., J. Biol. Chem. 193, 749 (1951). BERLINER, II. L., Federation Proc. 16, 219 (1956). BERLINER, D. L., AND SALHANICK, H. A., Anal. Chem. 28, 1608 (1956). SALHANICK, H. A., JONES, J. E., AND BERLINER, D. L., Federation Proc. 16, 160 (1956). SCHILLER, S., DORFMAN, R. I., AND MILLER, M., Endocrinology 36,355 (1945).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
71,
91-99
(1957)
Excretion of Steroids by the Newborn Infant’ Helen Strauss Hirs~h,~ David L. Berliner and Leo T. Samuels From the Departments of Biological Chemistry and Anatomy, University Utah College of Medicine, Salt Lake City, Utah Received
December
of
11, 1956
INTRODUCTION
Both chemical and histologic evidence indicates that there is a marked change in the steroid metabolism of the human being during the adjustment from intrauterine to independent existence. The involution of the fetal adrenal after birth is a well-documented phenomenon (1, 2) ; and Migeon et al. (3) have shown that the level of dehydroepiandrosterone in cord blood is higher than in the blood of the mother, but that it falls below measurable levels within a few days after birth. He concludes that the high levels of the steroid in the cord blood probably originate from the fetal rather than the maternal adrenal, and that the decrease is associated with the adrenal involution. Gardner (4, 5) has reported high levels of 17-ketosteroids in the plasma of newborn infants; these fell below measurable levels in a few days. Eagle (6) has described the presence of dehydroepiandrosterone and similar substances in the urine of premature babies, and Ulstrom and Doeden (7) have found a series of peaks when the Zimmermann reaction was applied to fractions from a column chromatogram prepared by a modification of the method of Dingemanse et al. (8). No consideration, however, has been given to the nonketonic steroids. In the present study the daily excretion of steroids during the first 10 days of life was investigated. The antimony trichloride reaction of Pincus (9) decreased progressively over the first 10 days. From the pooled nonketonic fractions androstane-3cr, 17/?-diol was crystallized and identified. 1 This work was supported in part by research grants from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service and from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 2 Present address : Bethesda, Maryland. 91
92
HIRSCH,
BERLINER
AND
SAMUELS
METHODS
A. Collection of Specimens Because of the difficulties in obtaining quantitative specimens of urine from newborn infants, it was decided to use the entire excreta as collected in the diapers. This would also allow the collection of steroids excreted via the intestinal tract and, since the gut of the newborn infant contains relatively few bacteria, errors due to the action of organisms would not be great. Cotton diapers which had been laundered in the hospital laundry were extracted with chloroform to eliminate the extraneous lipide material and then dried and sterilized. During each 24-hr. period each diaper, as soon as it was removed, was placed in a covered glass jar containing ethanol. The diapers were then allowed to stand in the ethanol for another 24 hr. with occasional agitation. At the end of this period the ethanol extract was drained as completely as possible from the cloths, and was replaced with butanol. This solvent was again left in contact with the diapers for another 24 hr. with occasional stirring and removed as before. A third extraction under the same conditions was then carried out with chloroform. The three extracts from each sample were combined and taken to dryness by vacuum distillation. The residue was dissolved in a small amount of chloroform and alcohol, 125 ml. of 4% sulfuric acid was added, and the mixture was hydrolyzed under reflux for 30 min. After cooling, the hydrolyzate was extracted four times with 50 ml. chloroform, the extracts were combined, and the solvent was removed by evaporation under vacuum. The residue was dissolved in ether and extracted with three portions of 2.5 N NaOH which was discarded. The ether layer was then washed once with an equal volume of 10% sulfuric acid and three times with equal volumes of distilled water. The extracts at the end of this procedure contained large amounts of lipide material and pigments, and removal of most of these impurities was essential for the subsequent analysis. The residue from the ether extract containing the neutral steroid fraction, therefore, was completely dissolved in a mixture of 2 parts of ether to 7 parts of acetone, and a small amount of saturated magnesium chloride solution in alcohol (approximately 30 drops/l0 ml. of ether-acetone mixture) was added. The sample was placed in the refrigerator for at least 8 hr. The solution was then filtered and the precipitate washed thoroughly with cold acetone. The ether-acetone solution was taken to dryness and the residue was distributed between 70% ethanol and pentane. The alcohol portion was extracted three times with equal volumes of pentane, and the combined pentane extract was then backwashed with one volume of 70% ethanol. The ethanol fractions were combined and the solvent was removed by evaporation; the pentane extract was discarded. The ethanol-soluble residue was then divided into ketonic and nonketonic fractions according to the procedure of Pincus and Pearlman (10) using Girard reagent T. Besides accomplishing the separation of ketonic and nonketonic steroids this procedure removed most of the remaining pigment from the samples. After evaporation of the ether, the ketonic and nonketonic residues usually consisted of small droplets of lipide material containing slight amounts of yellow pigment. Both the ketonic and nonketonic extracts were subjected to two color reactions: the Callow modification of the Zimmermann reaction (11,12) and the anti-
93
EXCRETION OF STEROIDS mony trichloride reaction of Pincus (9). Aliquots equal to one-fifth excretion were used for the color reactions unless otherwise specified. ing aliquots of each type of extract were then combined, and isolation steroids was attempted.
of the 24-hr. The remainof individual
RESULTS
After reactions with the Zimmermann reagent most ketonic extracts gave absorption curves which had maxima around 500 rnp while the nonketonic fractions showed only general decreasing absorption over the 400-700~pm range. As can be seen in Fig. 1, where the spectra for both ketonic and nonketonic fractions of three subjects are given, there was considerable background absorption, however, in all extracts. Typical results with the antimony trichloride reaction are shown in Fig. 2. All nonketonic fractions gave a green color with a maximum absorption around 600 rnp; this was absent from all ketonic samples. Of the various steroids tested by Pincus, the only nonketonic compound giving an absorption in this region was androstanediol; pregnanediol and cholesterol showed weak maxima at 680-690 rnp (9). Since reactions were obtained with both the ketonic and nonketonic Non-Ketonic Ketonic
400 FIG. 1. Absorption curves tracts from diaper collections Reaction.
500 WAVELENGTH
---
600
700
rnp
for ketonic and nonketonic fractions during the first 24 hr. after birth.
of three exZimmermann
94
HIRSCH,
BERLINER
AND
SAMUELS
Non-Ketonic Ketonic - - - - -
I
I
I
I
I
I
I
I
I
I
I
I
500
600 700 WAVELENGTH rnfi FIG. 2. Absorption curves for ketonic and nonketonic fractions of three extracts from diaper collections during the first 24 hr. after birth. SbCZ3Reaction.
extracts from excreta during the first 24 hr. of life, collections were continued over a IO-day period on two female infants. The results of the analyses, using the Allen correction for background absorption (13), are shown in Table I. Unfortunately, in the case of S.L. a number of samples were lost due to various factors, but the two series show the same general pattern. There is a tendency for the excretion of both types of compound to decrease. This is particularly striking in the case of the nonketonic substance which has practically disappeared by the tenth day. Because of the relatively strong reaction with antimony trichloride given by the early nonketonic fractions, the remaining portions of these fractions were pooled and an attempt was made to isolate and identify compounds therein. The pooled extract was separated into alcoholic and nonalcoholic fractions with succinic anhydride (10). Both the alcoholic and the nonalcoholic fractions were then chromatographed on aluminum oxide (Merck, according to Brockman). The chromatograms were developed with increasing proportions of benzene to petroleum ether, benzene, increasing concentrations of ethanol in benzene, and finally absolute ethanol. Most of the nonketonic, nonalcoholic fraction was eluted in the benzene-petroleum ether mixtures. No crystalline material was obtained
EXCRETION
OF
TABLE
of Androstane-$a,l7p-diol
Excretion
after 1
2 3 4 5 6 7 8 9 10
17-Kb
0.92 0.72 0.72 0.46” 1.19c
0.24 0.17 0.17 0.20” 0.40”
0.35 0.29 0.46 0.08
0.13 0.06 0.06
TABLE
of Nonketonic
Weight Fr%:?
of material
the First Baby
mg.
a Androstane-3cr, 17@-diol used as standard. b Dehydroepiandrosterone used as standard. c Probably portion of excretion from 4th day included
Chromatography
during
R.I.
mg. dial”
birth
I
and l’l-Ketosteroids Postnatal Days Baby
Days
95
STEROIDS
Alcoholic
Ten
S.L. mg.
mg. dioP
17-Kb
0.85 0.67
0.13 0.30
0.37 0.47
0.07 0.12
0.10
0.07
0.02
0.08
in collection
for 5th day.
II Fraction
put on the column:
Eluant
on Aluminum
Oxide
34.5 mg. Approx.
wt.
of fraction
m.
5 6 7 8 9 10 11 12
Benzene-petroleum ether 1: 1 Benzene Benzene 0.2 and 0.3’% alcohol in benzene 0.5, 0.7, 1 .O, and 2.0% alcohol in benzene 0.3% alcohol in benzene 4.0, 5.0, 7.0, and 10.0% alcohol in benzene 50.0y0 alcohol in benzene and absolute alcohol
2.6 1.2 3 .O (crystalline) 2.0 2.0 1.5 (crystalline) 2.0
in any fraction, and none of the fractions gave anything but background color, either with the Zimmermann reagent or antimony trichloride. As shown in Table II, two crystalline fractions were obtained from the nonketonic alcoholic material, one eluted with benzene (No. 7), and the other with 3% ethanol in benzene (No. 10). Fractions 7 and 10 were recrystallized from 70 % methanol. The supernatant solutions were drawn off and used for the antimony trichloride reaction, as well as the entire amounts of the other fractions. Fractions 5, 6, 12 and the supernatant from 7 gave only background color but fractions 8, 9, 11, and the super-
96
HIRSCH,
BERLINER
AND
SAMUELS
natant from 10 gave the typical green color observed in the diaper extracts; the supernatant from fraction 10 was particularly strong. The crystals of fraction 10 melted at 228°C. after recrystallization from dilute methanol. The acetate was prepared and melted at 160-162°C. These melting points correspond to that of androstane3cY) 17/?-diol, m.p. 223°C and its acetate, 162-163°C. (14). The free compound was then chromatographed on paper in the benzene-formamide system of Zaffaroni (15) where it had an R, of 0.21. This compound was eluted in methanol and divided into three aliquots for the following procedures: (a) chromogen determination with sulfuric acid (16)) (b) oxidation with chromic trioxide (17), and (c) acetylation with acetic anhydride-l-Cl4 (18). (a) The sulfuric acid chromogen showed maxima at 290, 340, and 410 rnp, identical with known androstane-&, 17/?-diol. (b) After oxidation the compound was chromatographed in the hexane-formamide system of Zaffaroni, where it showed an R, of 0.31, gave a positive Zimmermann reaction, and had a maximum of 290 mp in sulfuric acid, identical with known androstane-3,17-dione. (c) This aliquot was acetylated with acetic anhydride-l-C4 (specific activity, 2 mc./pmole) and pyridine overnight at room temperature. Acetylation was stopped by the addition of water, and the mixture was extracted with ethyl acetate. The ethyl acetate was evaporated, and at this time crystalline acetate originally prepared from the material isolated on the alumina column was added. The mixture was chromatographed on a paper immersed in a solution of methanol and formamide (2:s) in the heptane-formamide system at a room temperature of 4°C. The paper was dried and the compound identified by the microchemical isotopic technique described elsewhere (19). The compound had an R, of 0.77, identical with known androstan-3cy, 17p-diol-3,17-diacetate. A small amount of another radioactive contaminant with low polarity was found, which has not yet been identified. The radioactive androstan-3a, 17/3-dial-diacetate was hydrolyzed with 10 mg. of acylase using a 0.2 M phosphate buffer at pH 7.0 for 3 hr. at 37°C. The incubation was stopped by adding chloroform, and extracted. The chloroform extract was chromatographed in the benzene system, where the compound had an Rf of 0.23. This substance was further oxidized with chromic trioxide and run in the hexane-formamide system, where the compound had an Rf of 0.31, a positive Zimmermann reaction, and a maximum of 290 rnp in concentrated sulfuric acid, identical with
EXCRETION OF STEROIDS
IDENTIFICATION
97
OF ANDROSTAN-3~,17&diol.
FIG. 3. Method of identification of androstan-3a, 17&diol in chromatographic studies. (*) indicates position of CY4.
known androstan-3,17-dione. The evidence seems conclusive, therefore, that the crystalline nonketonic compound was androstane-3a, 17p-diol. The outline of the reactions used for identification is shown in Fig. 3. DISCUSSION
The evidence here presented indicates that one of the major (219 steroids excreted by the newborn infant is androstane-3cw,17@-diol. It appears to account for the reaction with antimony trichloride characteristic of the extracts during the first few days. Salhanick et al. found that radioactivity from testosterone-4-Cl4 did not cross the placental barrier, and that 4-androstene-3,17-dione was higher in cord blood than in the maternal circulation.3 Migeon et al. (3) also found that dehydroepiandrosterone conjugates were higher in the cord circulation. Apparently Cl9 compounds do not readily cross the placental barrier in either direction. Since the compound giving this reaction continues to be excreted during the first 10 days of life, it seems unlikely that it, or its precursor, entered the infant’s blood stream from the mother’s circulation. The more proba3 Salhanick, H. A., Jones, J. E., and Berliner, D. L., in preparation.
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HIRSCH, BERLINER AND SAMUELS
ble hypothesis seems to be that the disappearance of the compound is associated with the involution of the fetal zone of the adrenal cortex. What the precursor of androstanediol would be is uncertain. It may arise from complete reduction of the 4-androstene-3,17-dione found in cord blood (20). While androstanediol is perhaps a minor product of testosterone metabolism (21), it does not appear to be a major steroid in the urine of adult human beings. Its presence in infancy may represent a difference in metabolism. In the adult liver the conjugation process is so rapid that the major portion of the steroids are combined with the acids as soon as the ketone group on C-3 is reduced to an alcohol, and relatively little undergoes the further reduction at C-17. If conjugation mechanisms are not fully developed in the liver of the newborn, a greater portion of Cl9 steroids may be reduced to the diols before combination with glucuronic or other acids. It is also possible that the diol may have been present in the intestinal contents, a source which has not been well examined in the adult. Further work will be needed to determine its origin. ACKNOWLEDGMENT The advice and assistance of Dr. Harold L. Mason, Mayo Clinic, Rochester, Minnesota, during the isolation and recrystallization procedures is gratefully acknowledged. SUMMARY
The entire excreta from babies during the first few days after birth were extracted and analyzed for neutral steroids related to hormonal function. Material giving the Zimmermann reaction was found in the ketonic fraction, but no reaction with the antimony trichloride reaction of Pincus was obtained. The nonketonic fraction gave the antimony trichloride reaction. Both the ketonic Zimmermann-positive and the nonketonic antimony trichloride-positive substances decreased to very low levels during the first 10 days after birth. The material responsible for the color reaction in the nonketonic fraction was identified as androstane-3a!, 17fi-diol, probably a metabolic product of compounds formed in the fetal adrenal cortex. REFERENCES 1. ELLIOTT, T. R., AND ARMOUR, R. G., J. Pathol. Bacterial. 16, 481 (1911). 2. KERN, H., Deut. med. Wochschr. 37, 971 (1911). 3. MIGEON,~. J., KELLER, A. R., AND HOLMSTROM,E. G., Bull. Johns Hopkins Hosp. 97, 415 (1955).
EXCRETION
4. GARDNER, L. 5. GARDNER, L. 6. EAGLE, J. F., 7. ULSTROM, R. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21.
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I., J. Clin. Endocrinol. and Metabolism 13, 941 (1953). I., AND WALTON, R. L., Helv. Paediat. Acta 4, 311 (1954). Proc. Sot. Exptl. Biol. Med. 81, 571 (1952). A., AND DOEDEN, D., “Adrenal Function in Infants and Children,” Chap. 3, p. 31. Grune and Stratton, New York, 1955. DINGEMANSE, E., HUIS IN'T VELD, L., AND HARTOGH-KATZ, S., J. Clin. Endocrinol. and Metabolism 12, 66 (1952). PINCUS, G., Endocrinology 32, 176 (1943). PINCUS, G., AND PEARLMAN, W. H., Endocrinology 29, 415 (1941). CALLOW, ?J. H., CALLOW, R. K., AND EMI\IENS, C. W., Biochem. J. 32, 1312 (1938). ZIMMERMANN, W., Z. physiol. Chem. 233, 257 (1935). ALLEN, W. M., J. Clin. Endocrinol. 10, 71 (1950). RUZICKA, L., GOLDBERG, M. W., AND MEYER, J., Helv. Chim. Acta 18, 994 (1935). ZAFFARONI, A., Recent Progr. Hormone Research 8, 51 (1953). ZAFFARONI, A., J. Am. Chem. Sot. 72, 3828 (1950). ZAFFARONI, A., AND BURTON, R. B., J. Biol. Chem. 193, 749 (1951). BERLINER, II. L., Federation Proc. 16, 219 (1956). BERLINER, D. L., AND SALHANICK, H. A., Anal. Chem. 28, 1608 (1956). SALHANICK, H. A., JONES, J. E., AND BERLINER, D. L., Federation Proc. 16, 160 (1956). SCHILLER, S., DORFMAN, R. I., AND MILLER, M., Endocrinology 36,355 (1945).